Migration shift assays are useful methods to detect and quantify associations between biomolecules. A change in the retention time of a molecule in an electrophoretic or chromatographic assay, for example, can indicate the presence of a binding molecule. Binding can be specific, such as in the case of antibody-antigen interactions, or non-specific, such as the ionic attraction of a positively charged molecule to a negatively charged polymer. Interference from non-specific interactions of sample constituents in a migration shift assay should be minimized to prevent biasing of assay results.
Migration shift analysis on separation media can take many forms. For example, a change in retention time of a free nucleic acid can be observed by size exclusion chromatography (SEC) when it is bound to a protein. The SEC resin can include pores large enough for the free nucleic acid to enter, but too small for the nucleic acid/protein pair to enter. The nucleic acid/protein pair flows quickly in the volume around the SEC resin while the free nucleic acid flows more slowly through the total volume inside and outside of the resin. There is a “shift” in retention time between the free nucleic acid and the nucleic acid/protein pair. In addition, the size of a detected nucleic acid/protein peak can be interpreted to quantify the amount of the protein in the original sample. The presence of an interfering sample constituent can invalidate the results of a shift detection or quantitative assay.
In another example of migration shift analysis, free nucleic acid and a nucleic acid/protein pair can be separated by capillary electrophoresis (CE) through a separation media of a sieving polymer or gel which restricts the migration of large molecules, but allows freer flow of small molecules. In CE, an electroosmotic buffer flow is created by a direct electric current through a capillary tube. When current is applied, positively charged ions, and their associated solvating water molecules, migrate toward the cathode, creating an electroosmotic flow. A sample can be transported by this flow through a sieving polymer separation media in the lumen of a capillary tube to separate sample molecules by size for detection of a migration shift. The larger nucleic acid/protein pair will be entangled and impeded more than the free nucleic acid and thus exit the media later. A fluorescence or absorbance detector, for example, can monitor elution from the capillary tube to detect timed peaks which can be plotted on a chart to measure the time difference or “migration shift” between elution of the free nucleic acid and the nucleic acid/protein pair.
Recently, significant progress has been made in the application of microfluidics-based technologies which utilize microscale channel devices in various fields, for example, analysis of DNA, RNA, protein and metabolites. Advantages of such microfluidic technologies include reduction of reagent volume, higher resolution, shorter operation time, and easier solution handling.
A problem arises with some complex samples, such as samples derived from a human body such as blood or cell lysates, which can contain interfering constituents that bind non-specifically to assay components. For example, when the specific binding interaction of interest is the binding of a transcription factor to a specific target DNA sequence, a non-specific binding sample constituent can interfere with detection of the migration shift measurement. The interfering constituent can bind to the target DNA resulting in an insoluble complex that will not migrate in the separation media. The interfering constituent can create noisy background or false positive peaks by binding to the target DNA. In any case, non-specific binding of the target DNA can reduce the sensitivity and/or accuracy of the migration shift analysis.
Non-specific binding has been a problem in studies of DNA binding proteins. This problem was addressed in Brehm, BBRC 63: 24-31, 1975, where an anion exchange resin (QAE-Sephadex) was used to adsorb negatively charged blood serum proteins while washing away positively charged proteins that could non-specifically bind to the negatively charged DNA molecule. Adsorbed proteins were eluted from the QAE-Sephadex then applied to DNA-cellulose.
Proteins that bound to the DNA cellulose were identified as DNA binding proteins. Although this technique may have washed away some positively charged proteins that would have bound non-specifically to the DNA-cellulose, some of the proteins washed away were probably unidentified DNA binding proteins.
Instead of removing all positively charged proteins before a DNA binding assay, polyanion blocking agents can be added to assay solutions to minimize non-specific binding. In Carthew, et al., Cell 43: 439-448, 1985, poly dIdC was added to running buffers of a DNA binding gel electrophoresis migration shift assay to reduce the effect of proteins that bind non-specifically to the DNA. In such a strategy, poly-dIdC can compete with the target DNA for the non-specific DNA binding molecules, thereby reducing non-specific binding interference while enhancing the migration shift signal of any specifically bound proteins. Theoretically, DNA binding proteins specific for the target DNA can be detected, even if they are positively charged, since they can bind stronger to the target DNA, having both electrostatic and specific binding affinities. Although this blocking technology provides one way to enhance detection of DNA binding proteins, it fails to describe methods to enhance detection of migration shifts resulting from other types of specific binding interactions.
Migration shifts can be observed in other interactions of affinity molecules with analytes. Migration shifts can be observed, for example, when an antibody binds to an antigen, or when a polysaccharide binds to a lectin. However, chromatography or electrophoresis of these molecules often provides broad and poorly resolved peaks due to multiple conformations and unstable charge density in these molecules. The diversity of possible affinity molecule/analyte pairs can also require development of a special migration shift assay for each pair. These problems can be avoided if the affinity molecule is linked to a carrier polymer that is highly resolved in assays under a standard set of conditions. An example of technology using a carrier/affinity molecule conjugate is described, e.g., in Japanese Patent Application number WO 02/082083, “Method for Electrophoresis”, which is hereby incorporated by reference in its entirety. Although use of uniform carrier molecules for affinity molecules in migration shift analyses can improve resolution, a problem remains with interference due to non-specific binding.
A need therefore remains for methods to block the interference in migration shift assays, particularly in assays utilizing affinity molecule carriers. Migration shift assays of crude or complex samples can benefit from compositions, methods and apparatus that can block interference due to non-specific binding interactions with the migrating molecules. The present invention provides these and other features that will become apparent upon review of the following.
As mentioned above, migration shift assays provide very efficient separation and detection of the target analyte molecule (also referred to herein as the “objective substance” or “analyte of interest”). Moreover, the use of such migration shift assays in combination with microfluidic devices increases the efficacy of the assay. In order to increase the sensitivity of migration shift assays which use microfluidic devices, various methods for concentrating an objective substance (e.g., an analyte of interest) in a sample before applying the sample to a separation region of the device where the migration shift assay occurs, can be employed including, for example, (i) Field Amplification Sample Stacking (FASS), a method for concentrating the sample which utilizes the difference of electrical conductivities of a concentration domain and a separation domain (e.g., patent application Ser. No. 10/206,386 for “Microfluidic Methods, Devices and Systems for In Situ Material Concentration”, Weiss, D. J., Saunders, K., Lunte, C. E. Electrophoresis 2001, 22, 59-65; Britz-McKibbin, P., Bebault, G. M., Chen, D. D. Y. Anal Chem. 2000, 72, 1729-1735, Ross, D., Locascio, L. E. Anal Chem. 2002, 71, 5137-5145, the entire contents of which are incorporated by reference herein.), (ii) Field Amplification Sample Injection (FASI), a method for concentrating the sample by inserting a minute plug of water between the concentration domain and the separation domain in the FASS (e.g., “Field amplified sample injection in high-performance capillary electrophoresis”, Chien, R. L et al. J. Chromatogr. 1991, 559, 141-148, the entire contents of which are incorporated by reference herein), (iii) Isotachophoresis (ITP), a method for concentrating the sample which utilizes the difference of mobilities of ions in the domain sandwiched between a leading electrolyte solution and a trailing electrolyte solution (e.g., Everaerts, F. M., Geurts, M. Mikkers, F. E. P., Verheggen, T. P. E. M J Chromatagr. 1976, 119, 129-155; Mikkers, F. E. P., Everaerts, F. M., Peek, J. A. F. J. Chromatogr. 1979, 168, 293-315; and Mikkers, F. E. P., Everaerts, F. M., Peek, J. A. F. J. Chromatogr. 1979, 168, 317-332, Hirokawa, T, Okamoto, H. Ikuta, N., and Gas, B., “Optimization of Operational Modes for Transient Isotachophoresis Preconcentration-CZE,” Analytical Sciences 2001, Vol. 17 Supplement i185, the disclosures of which are incorporated in their entirety by reference herein), (iv) Isoelectric Focusing (IF), a concentration/separation method which utilizes the difference of isoelectric points between the substances (e.g., “High performance isoelectric focusing using capillary electrophoresis instrumentation”, Wehr T, et al. Am. Biotechnol. Lab. 1990, 8, 22, “Fast sand high-resolution analysis of human serum transferring by high-performance isoelectric focusing in capillaries”, Kilar F. et al., Electrophoresis 1989, 10, 23-29, the entire contents of which are incorporated by reference herein.), and (v) Solid Phase Extraction (SPE), a concentration/separation method which utilizes a specific interaction between a solid phase (e.g., a solid phase with a bound adsorbent such as a receptor) and an objective substance to adsorb the objective substance to the solid phase (e.g., “Microchip-based purification of DNA from Biological Samples”, Breadmore M. et al. Anal. Chem. 2003, 75, 1880-1886, the entire contents of which are incorporated by reference herein.).
However, when the objective substance is concentrated by using the above-mentioned conventional methods, unnecessary constituents (e.g., so-called “noise constituents” which interfere with the detection of the objective substance) are often concentrated simultaneously with the objective substance. As a result, when the sample concentrated by a conventional method is used as the sample for separation and detection, the detection sensitivity may be limited due to the increased background and noise levels. Furthermore, the conventional concentration methods which utilize electrophoresis such as FASS, ITP and IF cannot efficiently and highly concentrate an objective substance having a very large molecular weight or relatively low electrical charge.
That is, in the above-mentioned concentration methods, when the objective substance is assumed to be spherical, the mobility of the substance is shown by the following formula:μe=q/6 . . . r 
wherein μe is the electrophoretic mobility of a particular ion, q is the electrical charge of the ion, is the viscosity of a solution and r is a radius of the ion. As is clear from the above-mentioned formula, when the objective substance has a very large molecular weight and/or a small electrical charge, the electrophoretic mobility (μe) of the objective substance is reduced because r in the formula becomes large and/or q in the formula becomes small. Accordingly, when using such conventional concentration methods, it is difficult to highly concentrate an objective substance which has a very large molecular weight and/or a small electrical charge in a short time. Additionally, in the conventional concentration methods, in order to concentrate the objective substance in the sample, optimization of the reaction condition is often difficult, particularly when the objective substance coexists in a complex sample with various unnecessary interfering constituents (e.g., noise constituents) other than the objective substance which tend to get concentrated along with the objective substance. This is especially true in the case of serum samples used in the clinical diagnostics field, which samples contain a variety of substances to be measured with wide varieties of molecular weight and electrical charge distributions. As mentioned above, the development of a method to concentrate the objective substance efficiently and highly to detect the objective substance with high sensitivity and without increasing the background and noise levels, especially in connection with the use of microfluidic devices, would be advantageous. The present invention provides such methods and other features that will become apparent upon review of the following.